Jeanette Villanueva

Pathophysiological Trends During Withdrawal of Life Support: Implications for Organ Donation After Circulatory Death.

Background Donation after circulatory death (DCD) provides an alternative pathway to deceased organ transplantation. Although clinical DCD lung, liver, and kidney transplantation are well established, transplantation of hearts retrieved from DCD donors has reached clinical translation only recently. Progress has been limited by concern regarding the viability of DCD hearts. The aim of this study was to document the pathophysiological changes that occur in the heart and circulation during withdrawal of life (WLS) support. Methods In a porcine asphyxia model, we characterized the hemodynamic, volumetric, metabolic, biochemical, and endocrine changes after WLS for up to 40 minutes. Times to circulatory arrest and electrical asystole were recorded. Results After WLS, there was rapid onset of profound hypoxemia resulting in acute pulmonary hypertension and right ventricular distension. Concurrently, progressive systemic hypotension occurred with a fall in left atrial pressure and little change in left ventricular volume. Mean times to circulatory arrest and electrical asystole were 8 ± 1 and 16 ± 2 minutes, respectively. Hemodynamic changes were accompanied by a rapid fall in pH, and rise in blood lactate, troponin-T, and potassium. Plasma noradrenaline and adrenaline levels rose rapidly with dramatic increases in coronary sinus levels indicative of myocardial release. Conclusions These findings provide insight into the nature and tempo of the damaging events that occur in the heart and in particular the right ventricle during WLS, and give an indication of the limited timeframe for the implementation of potential postmortem interventions that could be applied to improve organ viability.

Improved heart function from older donors using pharmacologic conditioning strategies

BACKGROUND Hearts from older donors are increasingly being referred for transplantation. However, these hearts are more susceptible to ischemia-reperfusion injury (IRI), reflected in higher rates of primary graft dysfunction. We assessed a strategy of pharmacologic conditioning, supplementing Celsior (Genzyme, Naarden, The Netherlands) preservation solution with glyceryl trinitrate (GTN; Hospira Australia Pty, Ltd, Mulgrave, VIC, Australia), erythropoietin (EPO; Eprex; Janssen-Cilag, North Ryde, NSW, Australia), and zoniporide (ZON; Pfizer, Inc., Groton, CT), to protect older hearts against IRI and improve graft function. METHODS Wistar rats, aged 3, 12, and 18 months old, were used to represent adolescent, 30-year-old, and 45-year-old human donors, respectively. Animals were subjected to brain death (BD) and hearts stored for 6 hours at 2° to 3°C in Celsior or Celsior supplemented with GTN+EPO+ZON. Cardiac function and lactate dehydrogenase before and after storage were assessed during ex vivo perfusion. Western blots and histopathology were also analyzed. RESULTS After BD, 18-month hearts demonstrated impaired aortic flow, coronary flow, and cardiac output compared with 3-month hearts (p < 0.001 to p < 0.0001). After storage in Celsior, the recovery of aortic flow, coronary flow, and cardiac output in 18-month BD hearts was further impaired (p < 0.01 vs 3-month hearts). Percentage functional recovery of 18-month BD hearts stored in Celsior supplemented with GTN+EPO+ZON was equivalent to that of 3-month hearts and significantly improved compared with 18-month hearts stored in Celsior alone (p < 0.01 to p < 0.001), with reduced lactate dehydrogenase release (p < 0.01) and myocardial edema (p < 0.05) and elevated phosphorylated extracellular signal-related kinase 1/2 (p < 0.05) and phosphorylated Akt (p < 0.01). CONCLUSIONS Older hearts are more susceptible to IRI induced by BD and prolonged hypothermic storage. Supplemented Celsior activates cell survival signaling in older hearts, reduces IRI, and enhances donor heart preservation.

Ischaemic Tolerance and the Effect of Ageing Using a Rodent Donation after Circulatory Death (DCD) Model.

To investigate the effect of ageing on donor heart recovery after exposure to DCD withdrawal in a rodent model. Method Wistar rats (3, 12, 18 and 24 months) were anaesthetised and instrumented for invasive blood pressure and continuous heart rate/saturation monitoring using pulse oximetry. Circulatory death was induced by asphyxiation via tracheal ligation followed by injection of 500IU heparin via the renal vein. 3 month rats were exposed to 12 minutes warm ischaemia time (WIT). All other groups were exposed to 20 minutes WIT. At the end of the fixed WIT, hearts were flushed with 100mLs of celsior (C) or supplemented celsior(Cs) solution (containing erythropoietin, glyceryl trinitrate and zoniporide), followed by reperfusion on ex-vivo perfusion rig with oxygenated Krebs buffer at 37oc for 15 minutes followed by ‘working’ mode for 30mins. Aortic flow (AF), heart rate (HR) and pressure (MAP) were measured using PowerLab™; coronary flow (CF) was measured at 5 minute intervals. Results HR and MAP recovery were similar in all groups. CF and AF recovery are presented below: All results were corrected to heart weight[SVMHS1].CF was poorest in 3C group when compared to all other groups. AF recovery was superior in both 12mo groups when compared to other age groups (P<0.05); and all Cs groups show better AF recovery when compared to C only group. (P<0.05) Figure. No caption available. Conclusion These results suggest that myocardial tolerance to WIT increases through adolescence into adulthood then declines through the process of ageing. It is important to note, however, all age groups showed improved recovery after WIT with pharmacological supplementation.

Potential Limitation for the use of Cyclosporine A as a Cardioprotective Agent during Donor Heart Retrieval and Storage

Background The success of heart transplantation has allowed the consideration of older and sicker recipients for transplant but has necessitated consideration of hearts from “extended criteria” donors for transplant. We have successfully repurposed glyceryl trinitrate and erythropoietin for clinical use as additives to cardioplegic or protective cold flush solutions for routine clinical retrieval of marginal hearts from donors after both brain death or circulatory death (1, 2). Introduction of normothemic perfusion of the heart during transportation as an alternative to static cold storage has presented an opportunity to bolster the initial protective flush and the normothermic perfusate with extra cardioprotective agents. One such potential candidate is Cyclosporine A (CsA). It was shown to decrease cardiac ischemia reperfusion injury in pre-clinical models by minimizing mitochondrial permeability transition pore opening. The present study investigates the role of timing and duration of cardiac exposure to CsA during retrieval and storage on functional recovery and mechanisms of CsA action in a working rat heart model of donor heart preservation. Methods After measurement of baseline function, hearts were arrested and stored for 6h at 4°C in either Celsior® alone or Celsior®+CsA (0.2&mgr;M), then reperfused for 45min in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior®-alone stored hearts were exposed to 0.2&mgr;M CsA for the initial 15min (non-working period) or the full 45min period of reperfusion. Coronary effluent was collected pre- and post-storage for assessment of LDH release. Left ventricular free wall was snap-frozen after completion of each study for immunoblotting. Results Presence of CsA during storage or the first 15min reperfusion significantly improved functional recovery and significantly increased phospho-AMPKThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45min post-reperfusion recovered poorly with no phospho-AMPK increase, decreased mitochondrial cyctochrome c content and increased LDH release. Conclusions Inclusion of CsA at cardioplegia is cardioprotective. No extra benefit was gained by addition of CsA during the initial 15min reperfusion period. Presence of CsA for the full 45min reperfusion was toxic to the heart. Protective effects of CsA appear to be driven by activation of AMP associated protein kinase. References 1. Kumarasinghe G et al. Int J Transplant Res Med 2016, 2:018. 2. Dhital KK, et al. Lancet 2015; 385: 2585-91. National Health & Medical Research Council (program grant ID 1074386), Australia. St Vincents Clinical Foundation of Australia.

Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model

The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate. Hearts were arrested and stored in Celsior preservation solution supplemented with 0.2–40 μM dantrolene for 6 hours at 4°C, then reperfused (15 min Langendorff, 30 min working mode). In 3-month hearts, supplementation with 1 μM dantrolene significantly improved aortic flow and cardiac output compared to unsupplemented controls however lactate dehydrogenase (LDH) release and contraction bands were comparable. In contrast, 40 μM dantrolene-supplementation yielded poor cardiac recovery, increased post-reperfusion LDH but reduced contraction bands. All 3-month hearts stored in dantrolene displayed significantly reduced cleaved-caspase 3 intensities compared to controls. Analysis of cardioprotective signalling pathways showed no changes in AMPKα however dantrolene increased STAT3 and ERK1/2 signaling in a manner unrelated to functional recovery and AKT activity was reduced in 1 μM dantrolene-stored hearts. In contrast to 3-month hearts, no significant improvements were observed in the functional recovery of 12-month hearts following prolonged storage in 1 μM dantrolene. Conclusions: Dantrolene supplementation at 1 μM during hypothermic heart preservation improved functional recovery of young, but not older (12 month) hearts. Although the molecular mechanisms responsible for dantrolene-mediated cardioprotection are unclear, our studies show no correlation between improved functional recovery and SAFE and RISK pathway activation.

Pharmacological Conditioning of Brain Dead Donor Hearts with Erythropoietin and Glyceryl Trinitrate: Clinical Experience

Background: With the increasing success of heart transplantation, older and higher-risk donors and recipients are being accepted for transplantation. The risk of primary graft dysfunction (PGD) is thus increased. We investigated a ‘pharmacological conditioning’ strategy, where Celsior preservation solution supplemented with glyceryl trinitrate (GTN) and erythropoietin (EPO) was used for cardioplegia and hypothermic storage, and determined graft recovery and patient survival after cardiac transplantation.